It is proposed to use the cyclic AMP-dependent protein kinase of cultured S49 mouse lymphoma cells as a model system for stuyding mammalian gene regulation. S49 cell mutants resistant to cytolysis by dibutyryl cyclic AMP carry lesions affecting the kinase. Mutations in the structural gene for kinase regulatory subunit have been identified by two-dimensional polyacrylamide gel electrophoresis; cells carrying such mutations exhibit alterations in both function and level of expression of kinase holoenzyme. Other mutations appear to define a locus separate from kinase subunit genes which acts in a "trans" configuration to affect their regulation. Additional mutations affecting kinase expression will be isolated and characterized biochemically and genetically (by cell hybridization). Antisera will be prepared against kinase subunits and used in conjuction with two-dimensional gel electrophoresis to study mutational effects on structure, synthesis, degradation, and phosphorylation of these subunits. Mechanisms of kinase regulation will be probed further using specific inhibitors of protein synthesis and cell-free translation. Preliminary experiments will be performed to assess the feasibility of developing cell lines which overproduce kinase subunits and to develop means of mapping the structural and regulatory genes of the kinase system.